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Cloning, identification and functional characterization of two cytochrome P450 carotenoids hydroxylases from the diatom Phaeodactylum tricornutum. 2019

Hongli Cui, and Haotian Ma, and Yulin Cui, and Xiaoli Zhu, and Song Qin, and Runzhi Li
Institute of Molecular Agriculture and Bioenergy, Shanxi Agricultural University, Taigu 030801, China. Electronic address: cuihongli2005@163.com.

The diatom microalgal Phaeodactylum tricornutum accumulates a large amount of fucoxanthin. Carotenoids hydroxylases (CHYs) play key roles in fucoxanthin biosynthesis in diatoms. However, not any type of CHYs had been identified in P. tricornutum. In this study, two genes (designated Ptrcyp97b1 and Ptrcyp97b2) were cloned, identified and functionally characterized. They shared high sequence identity (50-94 %) with lutein deficient 1-like proteins from other eukaryotes. The typical catalytic active motifs of cytochrome P450s (CYP) were detected in the amino acid sequences of PtrCYP97B1 and PtrCYP97B2. The two genes were probably due to gene duplication. Ptrcyp97b1 and Ptrcyp97b2 transcriptional expression was up-regulated with distinct patterns under high light conditions. The metabolic profiles of the major carotenoids (β-carotene, zeaxanthin, diadinoxanthin, diatoxanthin and fucoxanthin) were determined based on the high performance liquid chromatography method. The fucoxanthin and diatoxanthin contents were increased, while the β-carotene content was decreased. By truncation of the N-terminal trans-membrane anchor or chloroplast transit peptide and addition of a 6 × His-tag, PtrCYP97B1 and PtrCYP97B2 were separately heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. Functional analysis showed that PrtCYP97B2 was able to catalyze the hydroxylation of the β-rings of β-carotene to produce zeaxanthin in β-carotene-accumulating E. coli BL21(DE3) cells. PtrCYP97B1 might have the ability to catalyze the hydroxylation of other substrates other than β-carotene. These results contribute to the further elucidation of xanthophyll biosynthesis in diatoms.

UI MeSH Term Description Entries
D002338 Carotenoids The general name for a group of fat-soluble pigments found in green, yellow, and leafy vegetables, and yellow fruits. They are aliphatic hydrocarbons containing 4 terpene subunits. Carotenes,Carotenoid,Tetraterpene Derivatives,Tetraterpenes,Carotene,Derivatives, Tetraterpene
D003001 Cloning, Molecular The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells. Molecular Cloning
D003577 Cytochrome P-450 Enzyme System A superfamily of hundreds of closely related HEMEPROTEINS found throughout the phylogenetic spectrum, from animals, plants, fungi, to bacteria. They include numerous complex monooxygenases (MIXED FUNCTION OXYGENASES). In animals, these P-450 enzymes serve two major functions: (1) biosynthesis of steroids, fatty acids, and bile acids; (2) metabolism of endogenous and a wide variety of exogenous substrates, such as toxins and drugs (BIOTRANSFORMATION). They are classified, according to their sequence similarities rather than functions, into CYP gene families (>40% homology) and subfamilies (>59% homology). For example, enzymes from the CYP1, CYP2, and CYP3 gene families are responsible for most drug metabolism. Cytochrome P-450,Cytochrome P-450 Enzyme,Cytochrome P-450-Dependent Monooxygenase,P-450 Enzyme,P450 Enzyme,CYP450 Family,CYP450 Superfamily,Cytochrome P-450 Enzymes,Cytochrome P-450 Families,Cytochrome P-450 Monooxygenase,Cytochrome P-450 Oxygenase,Cytochrome P-450 Superfamily,Cytochrome P450,Cytochrome P450 Superfamily,Cytochrome p450 Families,P-450 Enzymes,P450 Enzymes,Cytochrome P 450,Cytochrome P 450 Dependent Monooxygenase,Cytochrome P 450 Enzyme,Cytochrome P 450 Enzyme System,Cytochrome P 450 Enzymes,Cytochrome P 450 Families,Cytochrome P 450 Monooxygenase,Cytochrome P 450 Oxygenase,Cytochrome P 450 Superfamily,Enzyme, Cytochrome P-450,Enzyme, P-450,Enzyme, P450,Enzymes, Cytochrome P-450,Enzymes, P-450,Enzymes, P450,Monooxygenase, Cytochrome P-450,Monooxygenase, Cytochrome P-450-Dependent,P 450 Enzyme,P 450 Enzymes,P-450 Enzyme, Cytochrome,P-450 Enzymes, Cytochrome,Superfamily, CYP450,Superfamily, Cytochrome P-450,Superfamily, Cytochrome P450
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006900 Hydroxylation Placing of a hydroxyl group on a compound in a position where one did not exist before. (Stedman, 26th ed) Hydroxylations
D017377 Diatoms The common name for the phylum of microscopic unicellular STRAMENOPILES. Most are aquatic, being found in fresh, brackish, and salt water. Diatoms are noted for the symmetry and sculpturing of their siliceous cell walls. They account for 40% of PHYTOPLANKTON, but not all diatoms are planktonic. Bacillariophyta,Bacillariophytas,Diatom

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